TRPC channels blockade abolishes endotoxemic cardiac dysfunction by hampering intracellular inflammation and Ca2+ leakage

Intracellular Ca2+ dysregulation is a key marker in septic cardiac dysfunction; however, regulation of the classic Ca2+ regulatory modules cannot successfully abolish this symptom. Here we show that the knockout of transient receptor potential canonical (TRPC) channel isoforms TRPC1 and TRPC6 can ameliorate LPS-challenged heart failure and prolong survival in mice. The LPS-triggered Ca2+ release from the endoplasmic reticulum both in cardiomyocytes and macrophages is significantly inhibited by Trpc1 or Trpc6 knockout. Meanwhile, TRPC’s molecular partner — calmodulin — is uncoupled during Trpc1 or Trpc6 deficiency and binds to TLR4’s Pococurante site and atypical isoleucine-glutamine-like motif to block the inflammation cascade. Blocking the C-terminal CaM/IP3R binding domain in TRPC with chemical inhibitor could obstruct the Ca2+ leak and TLR4-mediated inflammation burst, demonstrating a cardioprotective effect in endotoxemia and polymicrobial sepsis. Our findings provide insight into the pathogenesis of endotoxemic cardiac dysfunction and suggest a novel approach for its treatment.


Supplementary methods
Measurement of mean arterial blood pressure (MAP). Mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Chronic indwelling catheters were placed in the femoral artery for MAP and heart rate measurement. Heparin sodium (100 IU/ml) solution was infused for the maintenance of catheter patency.
The catheter was connected to a pressure transducer and data were collected by a precalibrated PowerLab/4SP recording system (AD Instruments Pty Ltd., Bella Vista, New South Walles, Australia).
Following the completion of the surgical procedure, cardiovascular parameters were allowed to stabilize for 30 min. After LPS (50 mg/kg) was i.p. administered, MAP and heart rate were monitored over a 6-h period.
Histology and immunohistochemistry. The ventricular tissues were fixed in buffered formalin, dehydrated in graded ethanol, embedded in paraffin, and serially sectioned at 4-µm thickness. Standard hematoxylin and eosin staining and immunohistochemical staining with CCL3/MIP-1α (R&D systems, 1:50) antibody (Ab) were performed on these sections. The samples were examined and photographed with a Nikon Eclipse 80i microscope. All digital photographs were taken and measured in the same parameter setting. Image analysis was performed with ImageJ 1.49v (National Institutes of Health, Bethesda, MD, USA). The mean integrated optical density was assessed for manually cropped areas of uniform intensity in the field. The individual who analyzed the histologic samples was blinded to the treatment. Each data point is represented by 12 images from 4 animals.   Western blotting. Total proteins were extracted from cardiac tissues or cultured cells by RIPA buffer supplemented with protease inhibitor cocktail. Nuclear and cytoplasmic extracts were prepared using the nuclear and cytoplasmic protein extraction kit. Extracted proteins were quantified using the BCA protein assay kits. Western blotting was performed according to the standard protocol. In brief, equal amounts of proteins were denatured and separated by sodium dodecyl sulfate (SDS)-PAGE on 8% to 15% separating gels. The proteins were transferred to nitrocellulose membranes and the membranes were blocked in 5% BSA diluted in PBS-0.05% Tween-20 (PBS-T) for 2 h. Blots were incubated with primary Abs as follows: anti-rabbit secondary Ab (Abbkine, A21020, 1:10000), the membranes were rinsed in PBS-T, detected with ECL detection kit, and exposed in a Tanon-5200 Imaging System (Tanon Science and Technology Co., Ltd., Shanghai, China). Quantitative image analysis was performed with ImageJ 1.49v software. 5 Calcineurin activity analysis. Calcineurin activity was analyzed using Calcineurin Assay Kit according to the manufacturer's instructions. Briefly, ventricular tissue was homogenized in lysis buffer containing protease inhibitors on ice to extract soluble proteins. The protein samples were desalted using Bio-Spin 6 columns to remove free phosphates. The assay buffer containing RII phosphopeptide as the substrate for calcineurin was added. The samples were incubated for 20 min at room temperature. The reactions were stopped by adding Biomol Green reagent and then incubated at room temperature for 30 min. The absorbance was determined at 620 nm. Ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was used as the calcineurin inhibitor. Protein contents were determined using BCA assay kit.

RNA-seq and analysis.
The gene expression differences in the ventricles of WT, Trpc1 -/-, and Trpc6 -/mice were determined by RNA-seq 1 . DNA library preparation and sequencing were conducted by KangChen Bio-tech (Shanghai, China).

DNA library preparation and sequencing
Total RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA-Seq libraries were generated from 2 μg of total RNA using KAPA Stranded Total RNA LT Sample Prep Kit with Ribo-Zero Gold (Illumina, San Diego, CA) following the manufacturer's directions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94°C for 2 min. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5' exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA ligase. The ligated products were enriched by PCR amplification. The final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Sequencing was performed on an Illumina HiSeq 4000 in a 2×150 bp format. 6

Data analysis
Image analysis and base calling were performed using Solexa pipeline V1.8 (Off-Line Base Caller software, version 1.8). Trimmed reads (pass FastQC 0.11.5 filter) were aligned to the mouse reference genome (GenCode mm10) and the mouse transcriptome (GenCode mm10) using Hisat2 software (version 2.0.5). Transcriptional abundance estimation was completed via StingTie software (version 1.
Thereafter, tissue was minced into small pieces and subjected to enzymatic digestion with 450 U/mL collagenase I, 125 U/mL collagenase XI, 60 U/mL DNase I, and 60 U/mL hyaluronidase (Sigma-Aldrich) in b Representative immunofluorescent photomicrographs of TRPC1 or TRPC6 (green) localization.
Blue indicates cellular nuclei stained with DAPI. The colocalization is shown in yellow (white arrows) (n = 6 images from 3 male mice per group). Source data are provided as a Source Data file. Ca 2+ release in mice cardiomyocytes. a Trpc1 or Trpc6 knockout inhibits LPS-triggered intracellular Ca 2+ release in adult mice cardiomyocytes in Ca 2+ -containing and Ca 2+ -free extracellular solution, respectively.
14 Supplementary Fig. 4 The effects of LMWH treatment on the cardiac function and survivability of LPS-challenged mice. a The effects of LMWH treatment on the cardiac function of LPS-challenged mice.
Typical heart M-mode echocardiography still and ejection fraction are shown (mean ± SEM, n = 6 male mice per group). Statistical significance was determined using the one-way ANOVA with Tukey's multiple comparisons test. b Kaplan-Meier survival curves of LMWH-treatment on mice subjected to 50 mg/kg LPS (n = 10 male mice per group). Statistical significance was determined using the log-rank test. Source data are provided as a Source Data file. 15 Supplementary Fig. 5 Cluster heatmap of differential gene expression in the heart tissues using RNA-seq (n = 3 male mice per group).